Immunodetection of the toxic portion of Vip3A reveals differential temporal regulation of its secretion among Bacillus thuringiensis strains.

AIMS

To devise a protocol for heterologous expression and purification of a partial toxic portion of the Bacillus thuringiensis (Bt) vegetative insecticidal protein Vip3A and using it as an antigen for anti-Vip3A polyclonal antibody development. Also, to evaluate the regulation of Vip3A secretion into culture supernatants (SNs) of different Bt strains based on this antibody.

METHODS AND RESULTS

A primer pair was designed to amplify partially the toxic portion of the vip3A gene from the HD125 strain. The amplicon was cloned in expressing vector to produce a ~35 kDa peptide, which was HPLC-purified prior to rabbit immunizations. The serum containing the polyclonal anti-Vip3A antibody demonstrated a detection sensitivity of 0·4 ng mm for the antigen in slot-blot experiments. Seven Bt strains from different origins were assessed regarding their temporal secretion of Vip3A toxin. ELISA results showed a strain-specific temporal regulation of Vip3A secretion in culture for the temperate isolates, with no detection of the toxin for the tropical strains, even when the presence of the gene was confirmed by PCR and sequencing.

CONCLUSIONS

Conformational variation in the toxic portion of Vip3A may explain lack of its detection in the tropical strains. Isolates from the same subspecies display physiological variability in proteins' secretion into culture SNs, which can affect screening procedures for more effective strains/toxins.

SIGNIFICANCE AND IMPACT OF THE STUDY

Immunoassays based on the developed anti-Vip3A antibody can be useful in a variety of basic studies. This method can be also coupled with toxicity assays on target insects, for more efficient screening methods of novel Bt strains/toxins with biocontrol applicability.